The interaction of CD2 with its LFA-3 ligand expressed by autologous erythrocytes results in enhancement of B cell responses

The addition of autologous erythrocytes to unfractionated human mononuclear cell cultures results in enhancement of B cell responses to antigens and mitogens. This costimulating effect of red cells is abrogated by their preincubation with anti-LFA-3 monoclonal antibody. Preincubation of mononuclear cells with anti-CD2 monoclonal antibodies (anti-Leu 5b, OKT11, used singly) has a down-regulating effect on B cell activation and no enhancement of B cell responses is seen when red cells are added to anti-CD2-treated cultures. These results demonstrate a functional effect on B cells of the interaction between the CD2 molecule on T lymphocytes and its natural ligand, LFA-3. The precise mechanism by which this costimulating effect on B lymphocytes takes place is unclear. The study of T cell populations and T cell activation markers shows that the addition of erythrocytes causes a small but reproducible increase in the number of cells expressing the IL-2 receptor and the addition of IL-2 enhances the response of mononuclear cells to antigenic stimulation in the presence of erythrocytes. However, the supernatants of mononuclear cell cultures stimulated with pokeweed mitogen in the presence of autologous erythrocytes show decreased levels of IL-2, compared to supernatants of cells stimulated with pokeweed mitogen alone. The same supernatants show increased levels of interferon-gamma, but the addition of this lymphokine to cultures stimulated with pokeweed mitogen has no potentiating effect. It is possible that the effect of erythrocytes is mediated by other growth and/or differentiation factors, and additional studies will be required to clarify this point.     

Phorbol myristate acetate-activated keratinocytes stimulate proliferation of resting peripheral blood mononuclear lymphocytes via a MHC-independent, but protein kinase C- and intercellular adhesion molecule-1-dependent, mechanism.

Recent attention has focused on the role keratinocytes (KC) may play in the induction of T cell-mediated inflammatory responses in skin, particularly because KC, when activated by immunologic stimuli, express MHC class II Ag and secrete immunomodulatory cytokines. We tested the capacity of normal human KC that were stimulated with PMA to induce PBMC proliferation. PMA-treated, but not untreated, KC induced proliferation of allogeneic as well as autologous PBMC; in addition, when purified CD4+ or CD8+ T cells were used as responders, each subset proliferated. PBMC proliferation was not due to direct action of PMA on PBMC, nor to contamination of KC cultures with Langerhans cells (LC) or dermal APC. Pretreatment with different protein kinase C inhibitors abrogated the capacity of PMA-stimulated KC to induce proliferation. Paraformaldehyde-fixed PMA-KC stimulated PBMC proliferation, whereas supernatants from PMA-treated KC failed to do so, indicating that a membrane-associated activity on PMA-KC contributes to the induction of PBMC proliferation. PMA induced intercellular adhesion molecule-1 (ICAM-1) expression on KC; furthermore, mAb against ICAM-1 or against its ligand lymphocyte function-associated Ag (LFA-1) (CD11a/CD18) significantly, but incompletely, reduced the stimulatory capacity of PMA-treated KC, indicating that ICAM-1/LFA-1 interaction contributed to PBMC proliferation. IFN-gamma or TNF-alpha also induced ICAM-1 on KC, but these KC failed to stimulate proliferation, suggesting that PMA induces additional signals from KC, which act in concert with ICAM-1 to promote proliferation. Finally, mAb against HLA-ABC or HLA-DR did not inhibit proliferation. We conclude that PMA can activate KC to stimulate T cell proliferation in a MHC-independent fashion. This activation is mediated by protein kinase C and in part by the induction of ICAM-1 expression on KC.     

Role of CD8+ T cells in endogenous interleukin-10 secretion associated with visceral leishmaniasis

This study examined the role and source of endogenous interleukin-10 (IL) secretion in visceral leishmaniasis (VL). The amounts of endogenous and Leishmania specific IL-10 and interferon-gamma (IFN) secreted by peripheral blood mononuclear cells (PBMC) from VL patients were compared. The correlation coefficient between endogenous IL-10 secretion and Leishmania specific IFN-gamma was -0. 77, suggesting a major role for endogenous IL-10 secretion in VL. The effects of CD4+ and CD8+ T cell clones, isolated from a treated VL patient, on IL-10 secretion were assayed by mixing the clones with autologous, inactivated PBMC. The CD8+ clones mediated increased levels of IL-10 secretion in the presence of PBMC alone suggesting that CD8+ T cells may mediate endogenous IL-10 secretion.     

Anti-feline immunodeficiency virus (FIV) soluble factor(s) produced from antigen-stimulated feline CD8(+) T lymphocytes suppresses FIV replication

Feline immunodeficiency virus (FIV) causes AIDS-like symptoms in infected cats. Concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC) from chronically FIV strain PPR-infected cats readily expressed FIV. In contrast, when PBMC from these animals were stimulated with irradiated, autologous antigen-presenting cells (APC), at least a 10-fold drop in viral production was observed. In addition to FIV-specific cytotoxic T lymphocytes, anti-FIV activity was demonstrated in the cell-free supernatants of effector T lymphocytes stimulated with APC. The FIV-suppressive activity was induced from APC-stimulated PBMC of either FIV-infected or uninfected cats but not from ConA-stimulated PBMC. Suppression of FIV strain PPR replication was observed for both autologous and heterologous feline PBMC, was dose dependent, and demonstrated cross-reactivity and cell specificity. It was also demonstrated that the anti-FIV activity originated from CD8(+) T lymphocytes and was mediated by a noncytolytic mechanism.     

Stimulation of IFN-gamma, TNF-alpha, and TNF-beta secretion in IL-2-activated T cells: costimulatory roles for LFA-1, LFA-2, CD44, and CD45 molecules.

Lymphokine-activated killer (LAK) cells are peripheral blood lymphocytes (PBLs) that possess the ability to kill target cells in a non-major histocompatibility complex (MHC)-restricted manner. Both NK and T cells can be stimulated with interleukin-2 (IL-2) to become LAK cells. We previously reported that the interaction of LAK cells with tumor cells also induces the secretion of interferon-gamma (IFN-gamma). The NK subset of LAK (LAK-NK) cells is stimulated by tumor cells to secrete IFN-gamma in a non-MHC-restricted manner while the T cell subset of LAK (LAK-T) cells is stimulated to secrete IFN-gamma upon cross-linking of the T cell receptor (TCR)-CD3 complex. We here report that LAK-T cells stimulated with anti-CD3 mAbs and tumor cells secrete two additional cytokines, tumor necrosis factor-alpha (TNF-alpha) and TNF-beta/lymphotoxin (TNF-beta). In addition, we demonstrate that at least four other structurally unrelated molecules, in addition to the TCR-CD3 complex, on LAK-T cells participate in the stimulation of IFN-gamma, TNF-alpha, and TNF-beta production. These molecules are the lymphocyte function associated antigen-1 (LFA-1), lymphocyte function associated antigen-2 (LFA-2), CD44, and CD45. LFA-1 is an integrin, LFA-2 is a member of the immunoglobulin supergene family, CD44 is homologous to the cartilage link proteins, and CD45 is a tyrosine phosphatase. Ligands to three of these molecules have been identified; ICAM-1, LFA-3, and hyaluronic acid binding to LFA-1, LFA-2, and CD44, respectively. LFA-1, LFA-2, and CD44 are reported to function both as adhesion molecules and as costimulators in resting T cells. Our data suggest that these three molecules enhance IFN-gamma, TNF-alpha, and TNF-beta production by augmenting LAK-T cell to tumor cell adhesion and also by functioning as costimulators.     

Generation of activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration with interferon-gamma (IFN-gamma)

Activation of human peripheral blood mononuclear cells (PBMC) by interleukin 2 (IL 2) and the role of interferon-gamma (IFN-gamma) in the IL 2-induced activation were investigated. Activated killer (AK) cells against NK-resistant tumor cell lines were induced in the medium containing recombinant IL 2 (rIL 2) and autologous serum without any other stimulating agents. AK activity was induced by doses of rIL 2 as low as 3 U/ml, and reached a maximum at 10(3) U/ml. Incubation of PBMC with rIL 2 resulted in IFN-gamma production and augmented NK activity after 1 day of culture, and in induction of AK cells and proliferative response after 2 days of culture. These results suggested that endogenous IFN-gamma was required for rIL 2-induction of AK cells and proliferative response. To prove this, PBMC were cultured with rIL 2 and rIFN-gamma or were pretreated with rIFN-gamma before culture with rIL 2. Both rIFN-gamma treatments of PBMC augmented rIL 2-induced AK activity and proliferative response. rIL 2-induced IFN-gamma production was also enhanced by the rIFN-gamma pretreatment of PBMC. The addition of anti-IFN-gamma antibody to rIL 2 cultures abrogated the rIL 2-induced NK augmentation, AK generation, and proliferative response in proportion to the decreased amounts of endogenous IFN-gamma detectable in culture. rIFN-gamma and/or rIL 2 cultures of PBMC increased Tac antigen expression on cell surfaces as measured by flow cytometry. Enhanced Tac expression by rIL 2 was abrogated by adding anti-IFN-gamma antibody. These data indicate that: 1) AK generation and IFN-gamma production are mediated by IL 2, and 2) IFN-gamma production may be required for IL 2 induction of AK cells and proliferative response. These finding are consistent with the hypothesis that AK generation involves a collaboration between IL 2 and IFN-gamma, in which IL 2 stimulates PBMC to produce IFN-gamma, which in turn acts as a differentiation signal that may be involved in the IL 2-initiated AK generation and proliferative response.     

A 19-kDa human erythrocyte molecule H19 is involved in rosettes, present on nucleated cells, and required for T cell activation. Comparison of the roles of H19 and LFA-3 molecules in T cell activation

We have previously described a molecule on the SRBC surface which, in addition to the sheep equivalent of LFA-3, is involved in the process of rosette formation. It is made of a single, 14- to 19-kDa, polypeptide chain, and we termed this molecule S14. We have now identified, on the human E a molecule with a similar Mr albeit somewhat higher (19 kDa). The mAb against H19 efficiently block autologous or homologous rosettes by binding to human E. In addition, purified H19 molecules block rosettes made with human E and SRBC in a dose-dependent manner. The H19 molecule, like LFA-3, is not limited to the E surface, but is also present on many nucleated cells, including T cells and monocytes. Moreover H19, like LFA-3, is required for T cell activation: when we stimulated whole PBMC anti-H19 blocked [H3]TdR incorporation triggered via CD3, but not via CD2, in contrast to anti-LFA-3 that inhibited activation via both pathways. When a mixture of highly purified T-PBL and autologous paraformaldehyde fixed accessory cells (AC) was cultivated, anti-H19 or anti-LFA-3 mAb bound to AC blocked T cell proliferation. When high amounts of rIL-1 (100 U/ml) were added to purified T-PBL, no AC were required to sustain their proliferation upon stimulation via CD2, contrary to stimulation via CD3. When lower amounts of rIL-1 (10 U/ml) were used, fixed AC were still necessary to sustain proliferation via CD2. In this latter situation, anti-H19 mAb bound to AC could no longer inhibit T cell proliferation, whereas the anti-LFA-3 mAb was still inhibitory. When T-PBL were stimulated via CD2 in the presence of 100 U/ml of rIL-1, anti-LFA-3 did not induce any inhibition. Thus the inhibitory effect of anti-H19 and anti-LFA-3 mAb can both be accounted for by an effect on the AC molecules only, and not on the T cell molecules. F(ab')2 fragments of anti-H19 mAb produced the same pattern of inhibition as the whole Ig molecule, excluding an effect via the FcR. Moreover, purified preparations of the H19 molecules also produced inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell-dependent B cell activation

Anti-CD3 mAb can activate T cells to help in B cell activation as detected by late events, such as maturation of B cells into Ig-secreting cells (IgSC), or by early events, such as B cell surface expression of the activation marker CD23. Two different anti-CD2 mAb each inhibited anti-CD3-induced T cell-dependent B cell activation in a dose-dependent fashion. Neither irradiation of the T cells prior to culture nor depletion of CD8+ cells abrogated the inhibitory effects of anti-CD2 mAb. Despite the ability of these anti-CD2 mAb to inhibit anti-CD3-induced IL2 production, addition of exogenous IL2 to anti-CD2 mAb-containing cultures could not fully reverse the inhibitory effects on IgSC generation. Furthermore, addition of various combinations of IL1, IL2, IL4, and IL6 or crude PBMC or monocyte culture supernatants also could not reverse anti-CD2-driven inhibition. In T cell-depleted cultures, anti-CD2 mAb had no effect on the ability of IL4 to induce B cell CD23 expression, confirming that anti-CD2 mAb had no direct effect on B cells. However, in cultures containing T+ non-T cells, anti-CD2 mAb did partially inhibit IL4-induced B cell CD23 expression. Taken together, these observations demonstrate that certain CD2 ligands can modulate T cell-dependent B cell activation by a mechanism which, at least in part, involves a direct effect by the CD2 ligand on the T cell itself.     

Conditions for the generation of autoreactive human T cell clones: requirement for lymphokines other than interleukin 2 alone

In an attempt to determine whether factors other than interleukin (IL) 2 alone were necessary for the generation of autoreactive suppressive T cell clones, lymphocytes from HLA-Dw-matched allogeneic mixed leukocyte cultures (MLC) were propagated and cloned in purified IL 2, partially purified IL 2, conditioned medium (CM) from stimulated peripheral blood mononuclear cells (PBMC), or with purified IL 2 plus IL 3, IL 4, or interferon-gamma (IFN-gamma). Cloning efficiencies were very low in all cases (less than 10%) and of 48 clones tested only 6 were capable of autocrine proliferation after stimulation with autologous PBMC. Four of these clones were derived from populations expanded and cloned in CM, one from cultures with partially purified IL 2, and one with purified IL 2. All were CD4+ alpha/beta-T cell receptor. Their stimulation was blocked by anti-DR and broadly reactive MHC class II-specific monoclonal antibodies (mAb) but not by anti-DQ or anti-DP mAb. One clone was blocked exclusively by broad mAb but not by anti-DR, -DQ, or -DP mAb, and this was the only clone to suppress lymphocyte proliferation in allogeneic MLC, a property previously described for autoreactive clones derived under similar conditions detecting potentially novel lymphocyte activating determinants designated "DY." These results therefore suggest that DY-specific autoreactive suppressive clones are produced under these conditions only at a low frequency and that an unidentified factor other than IL 2, IL 3, IL 4, or IFN-gamma is involved in their generation.     

Effect of sex hormones on human T cell activation by concanavalin A

The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied. We show that neither 17 beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women. Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers. We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity. Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC. Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level. Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.     

Regulation of human B cell responsiveness by CD8+ T cells: differential effects of stimulation with monoclonal antibodies to CD3 and pokeweed mitogen

Previous studies have shown that human CD8-positive T cells activated by immobilized mAb to the CD3 complex have the capacity to support the generation of Ig secreting cells (ISC). The experiments reported here were undertaken to examine the nature of CD8+ T cell helper function in greater detail. CD8+ T cells that had been treated with mitomycin C (CD8+ mito) and stimulated by immobilized mAb to CD3 (64.1) provided help for the generation of ISC from resting B cells. By contrast, CD8+ mito did not support the generation of ISC in cultures stimulated by pokeweed mitogen (PWM). This could not be explained by differences in the production of IL2, since PWM and anti-CD3 induced comparable amounts of IL2 from CD8+ mito. In anti-CD3-stimulated cultures, CD8+ mito supported the generation of larger numbers of ISC when B cells were also activated with Staphylococcus aureus (SA). By contrast, in PWM-stimulated cultures, CD8+ mito did not provide help for SA-activated B cells. Rather, PWM-stimulated CD8+ mito appeared to suppress the generation of ISC induced by PWM-activated CD4+ mito or by SA + IL2, whereas anti-CD3-stimulated CD8+ mito did not. Only control CD8+ T cells, which were able to proliferate, exerted suppressive effects in anti-CD3-stimulated cultures. Examination of the functional capacities of a battery of CD8+ T cell clones indicated that the same clonal population of CD8+ cells could provide help or suppress responses when stimulated with anti-CD3 or PWM, respectively. The functional activities of CD8+ clones differed from those of fresh CD8+ cells. Thus, anti-CD3-stimulated CD8+ clones provided help for B cells regardless of whether they were treated with mitomycin C. Moreover, PWM stimulated suppression by CD8+ clones was abrogated by treating the clones with radiation or mitomycin C. These results indicate that helper T cell function is not limited to the CD4+ T cell population, but that help can also be provided by appropriately stimulated CD8+ T cells. Taken together, these results indicate that CD8+ T cells are not limited in their capacity to regulate B cell responses, but rather can provide positive or negative influences depending on the nature of the activating stimulus.     

Generation and characterization of continuous lines of CD8+ suppressor T lymphocytes

The ability to grow normal T lymphocytes in long term culture has advanced our understanding of T cell biology. The growth of CD4+ cell lines allowed a further evaluation and appreciation of functional subtypes within this group. Cytotoxic CD8+ T cells have been characterized as well. The routine and continuous culture of Ag-nonspecific CD8+ Ts cells has been difficult to achieve. We have found that CD8+ T cells that suppress T cell proliferation and lack cytotoxic activity against T cells can be routinely obtained from PWM or PHA-stimulated PBMC. Continuous culture of T cell blasts from PWM or PHA-stimulated PBMC resulted in the growth of CD4+ and CD8+ T cells. These lines developed suppressor cell activity within 7 days after stimulation with PWM and 3 to 4 wk after stimulation with PHA. Concomitant with the development of suppressor activity was the loss of CD4+ T cells resulting in homogeneous lines of CD8+ suppressor cells. These cell lines have been maintained in continuous culture for greater than 6 mo by addition of rIL-2 twice weekly and restimulation with feeder cells and PHA every 2 wk. Activity of these cell lines was relatively resistant to irradiation or treatment with mitomycin C. Both cell lines suppressed proliferation of autologous or heterologous CD4+ T cells stimulated with PWM, OKT3, or tetanus toxoid but failed to suppress proliferation of CD4+ T cells in a mixed lymphocyte reaction. CD4+ T cells stimulated with PWM produced equivalent amounts of IL-2 in the presence or absence of Ts cells but failed to express the IL-2R (TAC) on their surface in the presence of Ts cells. By contrast, CD4+ T cell lines or cytotoxic CD8+ T cell lines failed to suppress proliferation of CD4+ T cells. With these results we describe methods for the generation and continuous culture of Ag-nonspecific CD8+ Ts cells and define some of their properties. These cells lines should be helpful in further elucidating the functional and phenotypic repertoire of CD8+ Ts cells.     

Effect of glucocorticoids on the development of suppressive activity in human lymphocyte response to a polysaccharide purified from Candida albicans

The action of glucocorticoids on the proliferative response of human lymphocytes stimulated in vitro by MPPS has been investigated. The effect of Dex was dependent on the time of steroid addition to the cultures. Dex added at the beginning of the culture period inhibited, cell proliferation and IL 1/IL 2 synthesis, although not completely. However, a delayed addition of 24 to 48 hr resulted in an enhancing effect on cell proliferative responses that was maximal at day 4. The effect of Dex on T suppressor cell activity was then investigated. Dex added 1 day before the appearance of suppressor cells resulted in a marked decrease or disappearance of the suppressive activity. Moreover, primed T lymphocytes treated with Dex in the presence of exogenous IL 2 enhanced the proliferative responses of fresh autologous PBMC stimulated by MPPS. Taken together, our data suggest that glucocorticoids inhibit the differentiation of T suppressor cells and that IL 2 is unable to reverse this inhibitory effect.     

Release of lymphokines after infection with Epstein Barr virus in vitro. II. A monocyte-dependent inhibitor of interleukin 1 downregulates the production of interleukin 2 and interferon-gamma in rheumatoid arthritis

Epstein Barr virus (EBV)-infection of normal peripheral blood mononuclear cells (PBMC) in vitro induces IFN-alpha secretion from B cell and natural killer (NK) cell populations, and IFN-gamma secretion from T cells. IFN-gamma depends on prior elaboration of IL 2 and IL 1 that originates from monocytes and NK cells. PBMC from rheumatoid arthritis (RA) patients released moderately elevated levels of IFN-alpha (236 +/- 62 U/ml vs 168 +/- 34 in normals). In contrast, IFN-gamma was significantly lower in RA (88 +/- 34 U/ml vs 209 +/- 32) with an associated deficit in IL 2. A monocyte-dependent factor was shown to be responsible for this deficit, since monocyte depletion of RA cultures normalized the levels of IL 2 and IFN-gamma. Significantly lower levels of IL 1 activity were present in the supernatants of RA PBMC cultures as compared with normal cultures, and this was shown to be associated with presence of a nondialyzable IL 1 inhibitor. This inhibitor was capable of preventing the IL 1-dependent synthesis of IL 2 and IFN-gamma by normal PBMC. Exogenous IL 1 or IL 2 restored the deficient IFN-gamma secretion in RA PBMC. Thus, the deficient ability of RA lymphocytes to control EBV infection may be secondary to impairment of a monocyte-T cell interaction at the level of IL 1.     

Bacterial lipopolysaccharide-induced interferon-gamma production: roles of interleukin 1 and interleukin 2

Bacterial lipopolysaccharide (LPS) induced human peripheral blood mononuclear cells (PBMC) to produce interferon-gamma (IFN-gamma). Monocytes play a mandatory accessory role in this process, because purified T lymphocytes failed to produce IFN-gamma in response to LPS and the addition of 2% monocytes to T cell cultures resulted in an optimal LPS-induced IFN-gamma production. IFN-gamma production was abolished in the presence of monoclonal antibodies specific for HLA-DR antigen. Addition of exogenous interleukin 2 (IL 2) markedly enhanced IFN-gamma secretion by PBMC induced with LPS. The addition of anti-Tac antibody specific for IL 2 receptors abrogated IFN-gamma production, suggesting that an interaction of IL 2 with IL 2 receptors was involved. By using a specific antibody binding assay, LPS was shown to amplify IL 2 receptor expression on PBMC, whereas exogenous IL 2 showed only a negligible enhancing effect on the expression of its own receptors. Interleukin 1 (IL 1), a product of LPS-stimulated monocytes, potentiated IL 2-induced IFN-gamma production in the absence of LPS. Neither IL 1 nor IL 2 alone induced IFN-gamma production in purified T lymphocyte cultures. When added together, however, substantial levels of IFN-gamma were induced. An enhanced IL 2 receptor expression on T cells was also demonstrated as a result of the combined action of IL 1 and IL 2. These results suggest that induction of IFN-gamma by LPS is due mainly to the generation of IL 1 and an enhanced expression of IL 2 receptors.     

Sequential effects of prostaglandins and interferon-gamma on differentiation of CD8+ suppressor cells

We have previously demonstrated that differentiation of CD8+ Tp44- suppressor cells in pokeweed mitogen (PWM)-stimulated cultures requires soluble factors elaborated by CD4+ cells and monocytes, and that the monocyte signal for such differentiation can be replaced by prostaglandin E2 (PGE2). In this study, we explored the ability of interleukin 2 (IL 2) and interferon-gamma (IFN-gamma) to replace the CD4+ signal. When IL 2 or IFN-gamma was used at concentrations equivalent to those present in supernatants of PWM-pulsed cultures of CD4+ cells, no effect on differentiation of CD8+ cells was observed. However, a potent suppressor inducing activity was detected when IFN-gamma, but not IL 2, was mixed with supernatants derived from cultures of PWM-pulsed purified monocytes (M phi sup) or with 10(-8) M PGE2. Differentiated CD8+ suppressor cells (Ts) inhibited both PWM-stimulated proliferative response of CD4+ cells and immunoglobulin production by B cells. The signals mediated by the M phi sup or PGE2 and IFN-gamma were shown to act sequentially. That is, M phi sup or PGE2 was required initially, followed by an IFN-gamma-dependent differentiative step. These studies thus suggest a cascade of cellular interactions involving monocytes, CD4+ cells, and CD8+ Ts precursors that are required for the differentiation of CD8+ suppressor effector cells.     

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

CD2 (E receptor, LFA-3 receptor) and E2 molecules (Bernard, 1988) on human T lymphocytes, CD58 (LFA-3, lymphocyte function associated antigen 3) on human erythrocytes and S14,S42,S110-220 molecules (Bernard, 1987) of sheep erythrocytes are involved in rosette formation of human T lymphocytes with human or sheep erythrocytes. Rosette formation of human and macaque pan-T lymphocytes with tree shrew (Tupaia belangeri) red blood cells (TRBC) (TRBC rosette) has shown different physicochemical properties from that of rosette formation with sheep red blood cells (E rosette) (Ben, 1985). CD2, CD3/TCR complex, CD5, CD6, and CD7 are not involved in TRBC rosette formation (Zheng, 1990). In order to know whether E2, LFA-3,S14,S42 and S110-220 molecules are involved in TRBC rosette formation or human and macaque T lymphocytes, rosette inhibition and antigenic modulation or co-modulation were performed with relevant monoclonal antibodies (McAbs), and hemolytic assay and slide agglutination were also conducted. TRBC rosette formation of human and rhesus monkey PBL was not blocked by E2 McAb (inhibition rate 2.8% and 2.1%, respectively). In contrast, human E rosette formation was obviously blocked at inhibition rate of 49.8% and macaque E rosette formation was slightly inhibited (13.3%). The modulation or co-modulation of E2 molecule with E2 McAb did not affect human TRBC rosette formation. Similar results were shown in rosette formation inhibition of Jurkat cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

CTLA-4 blockade by a human MAb enhances the capacity of AML-derived DC to induce T-cell responses against AML cells in an autologous culture system

BACKGROUND: Cells from AML patients can differentiate into the phenotype of DC when cultured with GM-CSF and IL-4. Such cytokine-treated AML-derived DC (AML-DC) can stimulate autologous T cells. In this study we examined whether an anti-CTLA-4 MAb (MDX-010) could enhance the generation of autologous anti-AML T cells. METHODS: MAb MDX-010 was added to AML PBMC cultures in the presence of GM-CSF and IL-4, a previously reported AML-DC culture method of generating anti-AML T cells. T-cell activation and proliferation were examined thereafter. RESULTS: Addition of MDX-010 to GM-CSF/IL-4-conditioned AML-DC cultures induced a mean seven-fold increase in the numbers of autologous T cells compared with cultures without MDX-010 (P < 0.007). T cells stimulated by AML-DC with CTLA-4 blockade were significantly more cytotoxic towards autologous AML cells than those without MDX-010 (42 +/- 23% vs. 26 +/- 15%, E:T ratio of 20). T cells stimulated by AML-DC with CTLA-4 blockade had significantly greater proportions of T cells producing IFN-gamma in response to autologous AML cells than those cultured with AML-DC alone (10.7 +/- 4.7% vs. 4.5 +/- 2.4% for CD4+ IFN-gamma+ CD69+ and 9.8 +/- 4.1% vs. 4 +/- 2.1% for CD8+ IFN-gamma+ CD69+ with or without MDX-010; n = 7; P < 0.007, P < 0.003, respectively). DISCUSSION: CTLA-4 blockade enhances the activity and numbers of AML-reactive T cells that can be stimulated by autologous AML-DC and may enhance the efficacy of adoptive immunotherapy of AML.     

Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells

Human peripheral blood T cells were purified by a four-step procedure which included depletion of plastic-adherent cells, rosetting with sheep red blood cells, nylon wool passage, and treatment with mouse monoclonal antibodies to human Ia antigens plus complement. The purified T cells completely failed to proliferate to phytohemagglutinin (PHA). Bacterially derived recombinant human interleukin 2 (IL 2) reconstituted the proliferative response of resting T cells to PHA. The optimal concentration of IL 2 required was 100 to 200 U/ml. IL 2 alone caused no T cell proliferation. Both PHA and IL 2 needed to be present together for the proliferation of T cells to occur. Incubation of T cells with either PHA or IL 2 alone for up to 18 hr, followed by washing, then by the addition of the reciprocal reagent, resulted in no T cell proliferation. Expression of IL 2 receptors and of Ia antigens, as assessed by indirect immunofluorescent staining, revealed that both PHA and IL 2 needed to be present for Tac and Ia antigen expression by T cells. T cells incubated with PHA and IL 2 for 18 to 42 hr acquired responsiveness to IL 2. These T cells remained absolutely dependent on IL 2 for proliferation to occur. In contrast to T cells stimulated with PHA in the presence of monocytes, T cells stimulated with PHA and IL 2 released no detectable IL 2. The failure of IL 2 secretion was not caused by down-regulation of IL 2 production by IL 2 itself, because the addition of IL 2 to cultures of T cells stimulated with PHA in the presence of monocytes did not interfere with IL 2 production. These results indicate that IL 2 is a sufficient signal to induce the expression of its receptor in PHA-stimulated T cells and subsequent proliferation but is not sufficient to cause endogenous IL 2 release.     

Identification of a cord blood T cell subset of CD3+4-8-45R+ suppressing interleukin 2 production in the autologous mixed lymphocyte reaction and the mode of action of exogenous IL2 in the induction of IL2 production

As previously reported, the inability of cord blood T cells to produce IL2 in the autologous mixed lymphocyte reaction (AMLR) could be recovered by the treatment of stimulator non-T cells with interferon-gamma (IFN-gamma) and of the AMLR with exogenous IL2. In the present study, we showed that addition of untreated autologous cord blood T cells to the above-mentioned AMLR abrogated the IL2 production in a dose-dependent manner, suggesting active suppression by the untreated T cells because untreated cord blood T cells did not consume IL2. Suppressor activity was abrogated by the treatment of cord blood T cells with monoclonal anti-CD3 antibody plus complement or with monoclonal anti-CD45R (Leu 18) antibody, but not by the treatment with monoclonal anti-CD4 antibody and/or anti-CD8 antibody plus complement. These data showed that the cord blood suppressor T cells were CD3+4-8-45R+. This suppressor activity also disappeared by culturing with rIL2 for 8 hr. As the frequency of CD45R+ cord blood T cells was comparable to that of CD45R+ adult T cells and was minimally affected by the IL2 treatment, functional modulation of CD45R+ suppressor T cells by IL2 is suggested. Moreover, in spite of the inhibitory effect of anti-CD45R antibody on the suppressor activity, IL2 production was not induced merely by addition of anti-CD45R antibody directly to the responder cells in AMLR. Taken together, these data suggest the requirement of exogenous IL2 for IL2 production in that IL2-producing-precursor T cells themselves should be stimulated by IL2 in addition to the modulation of CD45R+ suppressor T cells by IL2.